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Ergot, caused by Claviceps purpurea sensu lato, is an economically important seed replacement disease of Kentucky bluegrass (Poa pratensis) and perennial ryegrass (Lolium perenne) seed crops. C. purpurea sensu stricto is considered the primary Claviceps species responsible, but genetic diversity and cryptic species within C. purpurea sensu lato have previously been reported. Fifty-six C. purpurea sensu lato isolates collected from P. pratensis (n = 21) and L. perenne (n = 35) in Oregon and Washington between 2010 and 2014 were characterized via random amplified polymorphic DNA (RAPD), partial internal transcribed spacer (ITS), β-tubulin and elongation factor-1α (EF-1α) sequences, conidial size, and ergot alkaloid chemotype. Based on RAPD analysis, seven isolates from P. pratensis and 33 isolates from L. perenne collected in Oregon corresponded to C. purpurea sensu stricto, and 13 isolates collected from P. pratensis in Washington and Oregon were identified as C. humidiphila. Partial ITS, β-tubulin, and EF-1α sequences identified 10 isolates from P. pratensis as C. humidiphila, and seven isolates from P. pratensis and 33 isolates from L. perenne were identified as C. purpurea sensu stricto. Several isolates generated ambiguous RAPD bands or sequences that prevented identification. Ergot alkaloid chemotype profiling found that ergocornine and its epimer were predominant in sclerotia from P. pratensis, whereas ergotamine and its epimer were most abundant in sclerotia from L. perenne. This study confirms the presence of the C. purpurea sensu lato species complex in the U.S. Pacific Northwest and suggests that more research is needed to characterize and mitigate Claviceps spp. infection of grass seed crops in North America.

Ergot, caused by

Lolitrem B is a potent tremorgen responsible for the syndrome known as “ryegrass staggers” in livestock consuming perennial ryegrass (Lolium perenne) infected with the endophyte fungus Epichloe festucae var. lolii. We evaluated clinical signs in cattle continuously fed endophyte-infected perennial ryegrass straw at four concentrations of lolitrem B (LB)/ergovaline (EV) (groups: I-2256/638, II-1554/373, III-1012/259 or IV

This case-control study adds to the growing body of knowledge on the medical, nutritional, and environmental factors associated with Nodding Syndrome (NS), a seizure disorder of children and adolescents in northern Uganda. Past research described a significant association between NS and prior history of measles infection, dependence on emergency food and, at head nodding onset, subsistence on moldy maize, which has the potential to harbor mycotoxins. We used LC-MS/MS to screen for current mycotoxin loads by evaluating nine analytes in urine samples from age-and-gender matched NS cases (n = 50) and Community Controls (CC, n = 50). The presence of the three mycotoxins identified in the screening was not significantly different between the two groups, so samples were combined to generate an overall view of exposure in this community during the study. Compared against subsequently run standards, α-zearalenol (43 ± 103 µg/L in 15 samples > limit of quantitation (LOQ); 0 (0/359) µg/L), T-2 toxin (39 ± 81 µg/L in 72 samples > LOQ; 0 (0/425) µg/L) and aflatoxin M1 (4 ± 10 µg/L in 15 samples > LOQ; 0 (0/45) µg/L) were detected and calculated as the average concentration ± SD; median (min/max). Ninety-five percent of the samples had at least one urinary mycotoxin; 87% were positive for two of the three compounds detected. While mycotoxin loads at NS onset years ago are and will remain unknown, this study showed that children with and without NS currently harbor foodborne mycotoxins, including those associated with maize.

Past research showed a strong linear correlation between levels of the mycotoxins lolitrem B (LB, a tremorgen) and ergovaline (EV, an ergot alkaloid and potent vasoconstrictor) in perennial ryegrass (PRG) forage. The purpose of this study was to characterize the excretion of these two compounds in beef cattle consuming PRG straw and to utilize liquid chromatography-tandem mass spectrometry to investigate the metabolism of LB and EV in excreta. Four groups of steers ( n = 6/group) were fed endophyte-infected PRG for 64 days (2256/638, 1554/373, 1012/259, or 247/100 μg/kg LB/EV). Concentrations of LB and EV in both PRG straw and feces showed a linear relationship to each other. Feces reflected a dose-response for both mycotoxins, with values increasing most rapidly through 21 days then plateauing. Urine contained no detectable level of either compound or the ergoline lysergic acid. Screening for metabolites showed oxidation and reduction biotransformations for both toxins, with additional conjugation products detected for ergovaline.

The Oregon State University Colleges of Veterinary Medicine and Agricultural Sciences instituted the Endophyte Service Laboratory to aid in diagnosing toxicity problems associated with cool-season grasses in livestock. The endophyte (Neotyphodium coenophalum) present in tall fescue (Festuca arundinacea) produces ergopeptine alkaloids, of which ergovaline is the molecule used to determine exposure and toxicity thresholds for the vasoconstrictive conditions "fescue foot" and "summer slump". Another vasoconstrictive syndrome, "ergotism," is caused by a parasitic fungus, Claviceps purpurea, and its primary toxin, ergotamine. "Ryegrass staggers" is a neurological condition that affects livestock consuming endophyte (Neotyphodium lolii)- infected perennial ryegrass (Lolium perenne) with high levels of lolitrem B. HPLC-fluorescent analytical methods for these mycotoxins are described and were used to determine threshold levels of toxicity for ergovaline and lolitrem B in cattle, sheep, horses, and camels. In addition, six clinical cases in cattle are presented to illustrate diagnosis of these three diseases.

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P

Recently, interest has developed for using essential oils from Western juniper (Juniperus occidentalis) foliage and Port Orford cedar (Chamaecyparis lawsoniana) heartwood in commercial products such as pest repellents and cosmetics. In order to gauge the relative toxicological risk that these oils pose to freshwater and marine organisms, the acute aquatic toxicity of these oils was evaluated using OPPTS guidelines to the cladoceran Daphnia magna, the rainbow trout Oncorhynchus mykiss and the green alga Selenastrum capricornutum. For western juniper foliage oil, no toxicity was exhibited toward D. magna or O. mykiss, even at 5.0 mg/L (the highest concentration tested and limit of solubility). For toxicity to S. capricornutum using algal cell density, the 72 and 96 h EC50 value was 1.7 mg/L and the no observable effect concentration (NOEC) was 0.63 mg/L. For Port Orford cedar heartwood oil, no toxicity was exhibited toward O. mykiss or S. capricornutum, even at 5.0 mg/L (the highest concentration tested and limit of solubility). The 48-h D. magna EC50 value was 1.9 mg/L; the NOEC values for algal cell density were 1.25 mg/L (72 h) and 0.63 mg/L (96 h). In summary, this study shows that western juniper foliage and Port Orford cedar heartwood oils demonstrate little to no risk to aquatic organisms.

Lippia alba (Mill.) N.E. Brown (Verbenaceae), also known as Lippia geminata HBK or Lantana alba (Mill.), is a shrub about 3 m tall. In Brazilian traditional medicine, it is commonly known as Erva-Cidreira, Cha do Tabuleiro, and Salsa Limao. The present work was conducted in order to compare the phytochemistry of the roots and leaves and to determine the acute toxicity of the aqueous extracts from the roots of L. alba. The main components in the leaf extract were citral, β-citral, carvone, hemimelitene, germacrene D, and d-limonene. The main components in the root extract were durene, carvone, patchoulane and hemimellitene. To our knowledge, this is the first report of a phytochemical analysis of the root extract from Lippia alba. The LD50 for the aqueous root extract was calculated to be 1156.25 mgkg−1. The animals presented stimulant and depressive behaviors, showing moderate toxicity, most likely due to the presence of iridoids and phenylpropanoids.

An enrichment of strictly anaerobic bacteria from ovine rumen fluid, which has previously been named L4M2, is known to detoxify animal hepatotoxins from the pyrrolizidine alkaloid family. These toxins are present in the tansy ragwort plant (Senecio jacobaea). These plants have been described in livestock animals' range forages in regions of the world such as the Northwest United States and South Africa. The bacterial enrichment was characterized by molecular cloning techniques and by the molecular fingerprinting technique of denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the enrichment revealed that the consortium is composed of no more than five putative bacterial species which associated to the Anaerovibrio, Desulfovibrio, Megasphaera, Prevotella, and Synergistes generas. These are all known to exist in the upper gastrointestinal tract of ruminant animals. This work improved upon previous attempts to characterize the consortium by obtaining nearly full-length ribosomal 16S rDNA sequences through cloning. The DGGE results were directly compared to the cloning data by polymerase chain reaction (PCR) amplifying eight phylogenetically representative clones and analyzing them by DGGE. Direct DGGE analysis of the enrichment displayed greater 16S diversity than the clone library used in this study, suggesting that at least one of the organisms present in the enrichment comprises less than 1% of the total cell population. These data will be used to further refine the enrichment in hopes of future use as a probiotic, which could be administered to animals challenged by the presence of tansy ragwort in their forage.

Quality assurance and quality control is the foundation of any diagnostic test. The two laboratories in the United States that use HPLC to quantitate endophyte toxins in tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne) are Oregon State University (OSU) and University of Missouri (MU). Japan, the major importer of grass straw has six new laboratories that will test agricultural imports for endophyte toxins. A quality assurance program was set up between the Japanese Ministry of Agriculture and the State of Oregon. The latter includes both OSU and the MU. All units are using an accurate crystalline standard and have exchanged "check" samples among themselves. To date OSU and MU have values that differ by 10%. OSU has identified a contaminating and coeluting peak as the cause of the differences. Both laboratories are changing to a Gemini column to rectify the differences. Japanese laboratories are in the process of evaluating their split check samples. Keywords: quality assurance, quality control, endophyte, tall fescue, perennial ryegrass

Ergovaline is an ergot alkaloid produced by the symbiotic endophyte Epichloe coenophiala, which can colonize varieties of the cool-season grass tall fescue (Festuca arundinacea). It is the principle toxicant responsible for the vasoconstrictive and reproductive sequelae seen in "fescue toxicosis" in livestock which consume forage exceeding the threshold of toxicity established for this compound. A new method for extraction of ergovaline from tall fescue seed and straw was optimized and validated, on the basis of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, with high-performance liquid chromatography-fluorescence detection. Fourteen extraction solvents were tested; 2.1 mM ammonium carbonate/acetonitrile (50/50, v/v) had the highest and most consistent recovery (91-101%). Linearity, limit of detection, limit of quantitation, accuracy,and intra- and interday precisions for tall fescue seed and straw were 100-3500 μg/kg, 37 and 30 μg/kg, 100 μg/kg, 98%, 3.0 and 1.6%, and 3.8 and 1.0%, respectively. When the currently used solid-phase extraction (SPE) and QuEChERS methods were applied to 17 tall fescue straw samples, there was good agreement (correlation coefficient 0.9978). The QuEChERS method achieved the goals of eliminating chlorinated solvents and developing a fast, efficient, reliable method for quantitating ergovaline in tall fescue forage that can be applied in a high-throughput food safety laboratory.

Mineral implications in health issues of captive rhinos have received much attention lately. This study was undertaken to establish reference values for the mineral status of rhinos. Serum/plasma samples of free-ranging black (Diceros bicornis) and white (Ceratotherium simum) rhinos and of captive black, white, Indian (Rhinoceros unicornis), and Sumatran (Dicerorhinus sumatrensis) rhinos, as well as liver tissue samples of captive black, white, and Indian rhinos were analyzed for mineral content. Circulating mineral levels of free-ranging animals were subject to variation according to region. In free-ranging animals, high molybdenum (Mo) values compared to horse normals were striking. Captive animals displayed even higher circulating Mo concentrations. The significance of iron (Fe) overload in captive specimens of the browsing rhinos (black and Sumatran) was confirmed. Hepatic Fe levels increased in blacks with age. Although this Fe overload is suspected to be linked with diets, the data indicate that this is not due solely to an excessive dietary Fe supply. Whereas the grazing species (white and Indian) had high liver copper (Cu) levels, the browsing species had low to marginal liver Cu concentrations. Liver concentrations of K, Mg, Co, and Mo increased with age in captive black rhinos. Additional findings include high circulating Se levels in all rhino species. Future research should be directed at investigating factors leading to high Fe levels, and at investigating Cu metabolism in captive rhinoceros species.

Objective—To compare hepatic metabolism of pyrrolizidine alkaloids (PAs) between sheep and cattle and elucidate the protective mechanism of sheep. Sample Population—Liver microsomes and cytosol from 8 sheep and 8 cattle. Procedure—The PA senecionine, senecionine N-oxide (nontoxic metabolite) and 6,7-dihydro-7-hydroxy- 1-hydroxymethyl-5H-pyrrolizine (DHP; toxic metabolite) were measured in microsomal incubations. The kcat (turnover number) was determined for DHP and N-oxide formation. Chemical and immunochemical inhibitors were used to assess the role of cytochrome P450s, flavin-containing monooxygenases (FMOs), and carboxylesterases in senecionine metabolism. The CYP3A, CYP2B, and FMO concentrations and activities were determined, in addition to the role of glutathione (GSH) in senecionine metabolism. Results—DHP concentration did not differ between species. Sheep formed more N-oxide, had higher N-oxide kcat, and metabolized senecionine faster than cattle. The P450 concentrations and isoforms had a large influence on DHP formation, whereas FMOs had a large influence on N-oxide formation. In cattle, CYP3A played a larger role in DHP formation than in sheep. FMO activity was greater in sheep than in cattle. Addition of GSH to in vitro microsomal incubations decreased DHP formation; addition of cytosol decreased N-oxide formation. Conclusions and Clinical Relevance—Hepatic metabolism differences alone do not account for the variation in susceptibility seen between these species. Rather, increased ruminal metabolism in sheep appears to be an important protective mechanism, with hepatic enzymes providing a secondary means to degrade any PAs that are absorbed from the rumen. (Am J Vet Res 2004;65:1563–1572)

The chemical composition of essential oils extracted from Xylopia frutescens Aubl. (Annonaceae) fruit (collected in Pernambuco, Brazil) was examined by TLC and GC/MS. TLC analysis detected compounds from the flavonoid and terpenoid groups. GC/MS detected 23 terpenoid compounds, in which the principal components were germacrene D (24.2%), linalool (12.1%), β-pinene (8.0%), cis-sabinene hydrate (7.9%), trans-pinocarveol (7.8%), α-copaene (7.0%) and limonene (5.6%). Other constituents found were sabinene, m-cymene, 1,8-cineole, β-ocimene, perillene, cis-sabinol, α-campholenal, isopinocamphone, terpinen-4-ol, verbenone, trans-carveol, carvone, perillyl alcohol, α-cubebene, β-cadinene and elemol. Most of these compounds have not been detected in fruit extracts of X frutescens before; therefore this study represents the most comprehensive analysis to date of the chemical components in the fruits of this plant.

A method for the routine determination of the necine base retronecine from biological matrices is described, using gas chromatography for quantification. The biological matrices studied included blended sheep whole rumen fluid and bacterial growth media. The structurally similar compound 2,6-dimethoxypyridine was utilized as an internal standard. Prior to gas chromatography, the bis(heptafluorobutyrate) derivatives of both compounds were formed. The relative percent recoveries of retronecine and the internal standard were 73% and 82%, respectively. The detection limit of retronecine in blended whole rumen fluid was found to be 0.09 μg/mL, and 0.02 μg/mL in bacterial growth media. The precision of the peak area ratio (retronecine to internal standard) was 10% from blended whole rumen fluid, and 14% from bacterial growth media. This method was used to analyze samples from viable cultures incubated with retronecine.

The ability of ruminal microorganisms to degrade octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (high melting explosive, HMX) as consortia from whole rumen fluid (WRF), and individually as 23 commercially available ruminal strains, was compared under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography, followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) for delineation of the metabolic pathway. In WRF, 30 μM HMX was degraded to 5 μM HMX within 24 h. Metabolites consistent with m/z 149, 193 and 229 were present throughout the incubation period. We propose that peaks with an m/z of 149 and 193 are arrived at through reduction of HMX to nitroso or hydroxylamino intermediates, then direct enzymatic ring cleavage to produce these HMX derivatives. Possible structures of m/z 229 are still being investigated and require further LC-MS/MS analysis. None of the 23 ruminal strains tested were able to degrade HMX as a pure culture when grown in either a low carbon or low nitrogen basal medium over 120 h. We conclude that microorganisms from the rumen, while sometimes capable as individuals in the bioremediation of other explosives, excel as a community in the case of HMX breakdown.

Fescue toxicosis and perennial ryegrass staggers are two of the most common toxic plant diseases plaguing livestock in the United States, and result from consumption of forage containing the endophyte-produced mycotoxins ergovaline and lysergic acid (fescue toxicosis) and lolitrem B (ryegrass staggers). Our group has developed analytical assays for detecting these compounds, which serve a dual purpose (1) high-performance liquid chromatography-fluorescence assays are used to measure these compounds in feed material in order to promote “safe feed” through diagnostic testing in a service laboratory environment, and (2) highly sensitive and specific liquid chromatography-tandem mass spectrometry assays are utilized to study the fate and metabolism of these compounds in a diversity of livestock matrices so that a more refined understanding as to the etiology of the diseases these compounds cause can be achieved. A discussion applying these techniques to both current and anticipated studies is given, with an emphasis on impacts to trade and food safety regulation.

In this study, the rumen was assessed for its potential to detoxify RDX using molecular microbial ecology as well as analytical chemistry techniques. Results indicated significant loss (P

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P0.001) difference in the microbial population between the two ruminants irrespective of the primers used in the analysis. Qualitative comparison of the data provides evidence for the presence of similar phyla profiles between the 16S rRNA gene and the newly developed rpoB primers. A comparison between the two rpoB-based primer sets (old and new) showed that the old rpoB-based primers failed to amplify phylum Bacteroidetes (a common phylum in the rumen) in both bovine and ovine rumen samples. The old and new rpoB-DGGE-based primers amplified a large number of clones belonging to phylum Proteobacteria, providing a useful insight into the microbial structure of the rumen. ChaoI, ACE, Simpson, and Shannon-Weaver index analysis estimated the bovine rumen to be more diverse than the ovine rumen for all three primer sets. These results provide a new insight into the community structure among ruminants using the newly developed primers in this study.

This study investigated the fate and uptake of [(14)C]-TNT from soil into orchardgrass (Dactylis glomerata), perennial ryegrass (Lolium perenne), and tall fescue (Festuca arundinacea) over a one year period in a greenhouse-controlled environment. Pots (n = 4 for each grass, containing 10 mg cold TNT/kg soil + 1.2 mg [(14)C]-TNT/kg soil and controls with no TNT) were exposed to light and temperature conditions typical of June at 45 degrees N for 369 days. Three plant harvests were made (63, 181, and 369 days), and soil and plant materials were monitored for [(14)C]-TNT and metabolite concentrations. The 11.2 mg/kg TNT dose was not phytotoxic to the plant species tested. Continual uptake of TNT into grass blades was observed over the one-year period, with a total accumulation of 1.3%, 0.9%, and 0.8% of the initial soil [(14)C]-TNT dose for orchard grass, perennial ryegrass, and tall fescue, respectively. All [(14)C]-TNT residue in plant material was incorporated as bound residue. At final harvest, radioactivity was concentrated most highly in the root > crown > blade for all species. Soil TNT was gradually reduced to aminodinitro-toluenes and then further to an unidentified metabolite(s). Overall, orchardgrass appeared to be the most efficient species at taking up TNT.

Intravenous dimethyl sulfoxide (DMSO) has been utilized as a treatment for a number of acute inflammatory conditions and for cerebral edema following trauma in experimental and clinical cases. 2,5,9, 11,12,15,16,18,20.2t.23,2s In humans, DMSO at 1 g/kg, has been found to be more effective than mannitol, urea, cortisone or barbiturates in treating severe head trauma. TM This treatment is often the most economical and effective way of treating brain edema in acute equine neurological diseases, z In the horse, recommended intravenous dosages range from 0.1 g/kg to 4 g/kg in varying concentrations of up to 40% DMSO. l'2'r'1s'21 While the most commonly used dose is 1 g/kg 1,2,19,~ the adverse effects of DMSO given too rapidly at any dose have not been well documented in horses. Adverse effects relative to DMSO have been reported in human beings and a number of animal species. 4,8 These include intravascular hemolysis, hemoglobinuria, hyperosmolality, and transient increase in respiratory rate. Clinical signs associated with near lethal doses in experimental animals were spontaneous motor activity, tremors, myasthenia

Tall fescue toxicosis and ergot alkaloids cost U.S. livestock producers approximately one billion dollars in annual livestock production loss annually. Ergovaline (EV) is the tall fescue alkaloid primarily responsible for clinical disease in livestock. Since native ruminal microorganisms have not been attributed to the detoxification of EV, finding detoxifying microbes from other environments is desirable. One possible source for potential microorganisms that can degrade EV is the anaerobic gut of the earthworm, Eisenia fetida. This study describes a comparative microbial analysis of earthworm digestive tracts receiving 10,000 ppb EV (E+ treatment) when compared with a control treatment with no detectable amounts of EV (E− treatment). An HPLC assay determined a 25% loss of EV from the E+ treatment was microbial in nature. A community microbiomic approach of constructing 16S-rRNA gene clone libraries was used to compare the microbes affected by the two treatments. RDPII tools such as Classifier and Libcompare were used in the analysis of 16S sequences. DOTUR analysis was used to examine the richness and diversity of the two microbial populations in these experiments. The results indicate there are few significant differences in the microbial community structure between the two microbiomes.

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with

Ergot alkaloids are mycotoxins generated by grass and grain pathogens such as Claviceps, for example. Ergot alkaloid-poisoning syndromes, such as tall fescue toxicosis from endophyte-infected tall fescue grass, are important veterinary problems for cattle, horses, sheep, pigs and chickens, with consequent impact on food, meat and dairy industries. Damage to livestock is of the order of a billion dollars a year in the United States alone. HPLC with UV and fluorescence detection are the predominant means of ergot alkaloid determination, with focus on quantitation of the marker compound ergovaline, although ELISA methods are undergoing investigation. These techniques are excellent for rapid detection, but of poor specificity in defining new or poorly characterized ergot alkaloids and related compounds. This paper demonstrates the facility of using electrospray(+) mass spectrometry with multiple reaction monitoring (MRM) detection during chromatographic examination of ergot alkaloid standards of lysergic acid, lysergol, ergonovine, ergovaline, ergotamine, ergocornine, ergocryptine and ergocrystine by HPLC. Ergoline-8 position epimers could be separated on the gradient HPLC system for ergocornine, ergocrystine and ergonovine and appeared as shoulders for ergotamine and ergovaline; epimers generally showed different patterns of relative intensity for specific MRM transitions. There was reasonable correspondence between retention of standards on the 2-mm ESI(+)MS phenyl-hexyl-based reverse phase column and those on the 4-mm C18-based column. Since up to 10% of clinical cases involving toxin exposure display unidentified chromatographic peaks, 11 samples of feed components associated with such cases were studied with developed MRM methods to attempt elucidation of crucial components if possible. Ergotamine appeared in all, ergovaline appeared in five and ergocornine appeared in six; ergonovine, ergocryptine, ergocrystine and lysergol also appeared in several. In addition, molecular weights of compounds newly revealed by mass spectrometry suggested ergosine, ergostine and ergoptine in four samples, for which standards were not available. Dehydrated products of ergotamine, ergocrystine and ergocornine were discovered, along with dihydrogenated ergocrystine and ergocryptine in seven of the samples, and the issue was raised as to whether dehydration was strictly an instrument-derived artifact. Finally, five of the samples, along with fescue seed standard, evidenced one or more of 14 new ergot alkaloids ranging in size from 381 to 611 molecular weight and with key mass spectral characteristics of ergot alkaloids, specifically the pair of peaks m/z 223 and 208, corresponding to the ergoline ring system and its demethylated variant, respectively. It is anticipated that findings such as these will provide impetus to future development of analytical methodology for these heretofore relatively rare ergot alkaloid species.

Objective—To determine whether iontophoretic administration of dexamethasone to horses results in detectable concentrations in synovial fluid, plasma, and urine. Animals—6 adult mares. Procedure—Iontophoresis was used to administer dexamethasone. Treatments (4 mA for 20 minutes) were administered to a tarsocrural joint of each mare. The drug electrode contained 3 ml of dexamethasone sodium phosphate at a concentration of 4 or 10 mg/ml. Samples of synovial fluid, blood, and urine were obtained before and 0.5, 4, 8, and 24 hours after each treatment. All samples were tested for dexamethasone using an ELISA. Synovial fluid also was evaluated for dexamethasone, using high-performance liquid chromatography. Results—The lower and upper limits of detection for dexamethasone in synovial fluid with the ELISA were 0.21 and 1.5 ng/ml, respectively. Dexamethasone administered at a concentration of 10 mg/ml was detected by the ELISA in synovial fluid of 5 mares from 0.5 to 24 hours and in urine of 4 mares from 0.5 to 8 hours after each treatment, but it was not detected in plasma. Mean synovial fluid concentration of dexamethasone was 1.01 ng/ml. Dexamethasone administered at a concentration of 4 mg/ml was detected by the ELISA in urine of 2 mares at 0.5 and 4 hours after treatment, but it was not detected in synovial fluid or plasma. Conclusion and Clinical Relevance—Iontophoresis cannot be considered an effective method for delivery of dexamethasone to synovial fluid of horses, because drug concentrations achieved in this study were less than therapeutic concentrations. (Am J Vet Res 2002;63:11–14)

Objective—To determine the serum concentrations and sedative effects of fentanyl after transdermal administration at 3 dosages in llamas. Animals—9 healthy adult female llamas (mean age, 8 ± 3 years; mean weight, 150 ± 18 kg). Procedure—Llamas were allocated to 1 of 3 groups (3 llamas/group). Fentanyl patches (each providing transdermal delivery of 75 µg of fentanyl/h) were placed on shaved areas of the antebrachium of all llamas. In group 1, llamas were treated with 1 patch (anticipated fentanyl dosage, 75 µg/h). In group 2, llamas were treated with 2 patches (anticipated fentanyl dosage, 150 µg/h). In group 3, llamas were treated with 4 patches (anticipated fentanyl dosage, 300 µg/h). For each llama, the degree of sedation was assessed by use of a subjective scoring system and a blood sample was collected for determination of serum fentanyl concentration at 12, 24, 36, 48, 60, and 72 hours after patch placement. Results—Following the placement of 4 patches, mean ± SD serum fentanyl concentration in group 3 llamas reached 0.3 ± 0.08 ng/mL within 12 hours. This concentration was sustained for 72 hours. In group 2, application of 2 patches provided inconsistent results; in group 1, application of 1 patch rarely provided measurable serum fentanyl concentrations. No llamas became sedated at any time. Conclusions and Clinical Relevance—Results suggest that application of four 75 µg/h fentanyl patches provides consistent, sustained serum fentanyl concentrations without sedation in llamas. However, the serum concentration of fentanyl that provides analgesia in llamas is not known. (Am J Vet Res 2005;66:907–909)

Tall fescue toxicosis and other maladies in livestock result from the ingestion of vasoconstrictive ergot alkaloids produced by fungal endophytes associated symbiotically with the grass. In order to facilitate future analyses of grass extracts considered responsible for outbreak of related livestock diseases, we examined the electrospray ionization mass spectra of specific ergot alkaloids under conditions that permit protonation. Our purposes were both to record the spectra with interpretation of mechanisms of fragmentation and to derive commonalities that would allow the prediction of mass spectra of related compounds for which standards were not readily available. With [M + H](+) values in parentheses, water-insoluble lysergic acid peptide ergot derivatives ergovaline (m/z 534), ergotamine (m/z 582), ergocornine (m/z 562), ergocryptine (m/z 576) and ergocrystine (m/z 610) exhibited a consistent loss of water (-18 u) from the C-12' alpha-hydroxy functionality. Of this group, ergovaline and ergotamine generated an m/z 320 fragment deriving from cleavage of ring E amide and ether functions with retention of the peptide ring system methyl group. Ergocornine, ergocryptine and ergocrystine similarly formed an m/z 348 fragment with retention of isopropyl. These assignments were supported by the lack of similar fragments from the water-soluble ergot ergonovine, which lacks a peptide ring system. Clavine-type ergot alkaloids lysergic acid and lysergol lack any substituents beyond simple ones directly on the C-8 position and, similarly to ergonovine, lack significant fragments at m/z 268, 251 and 225 shared by the peptide ergot alkaloids.

The essential oil extracts of western juniper oil (Juniperus occidentalis) and Port-Orford-cedar oil (Chamaecyparis lawsoniana) were evaluated for possible dermal toxic effects on mice and rabbits. Mice were tested for their response to both extracts utilizing a local lymph node assay. Western juniper oil extract at 0.5% and 5% concentrations did not show a stimulation index (SI) greater than normal (3.0); however, a 50% concentration did show a positive response at 3.3. Port-Orford-cedar oil extract did not show a positive response at concentrations of 0.5%, 5% or 50%. An acute dermal irritation study using rabbits had a primary irritation index (PII) of 3.3 with 100% Port-Orford-cedar oil extract. This was reduced to a PII of 0.625 when diluted 1:1 with olive oil. Undiluted western juniper oil extract had a PII score of 2.7. While a 5.0% solution had a PII score of 0.3, a 0.5% solution of western juniper oil was a non-irritant. It would appear that animals bedded on wood shavings have contact with essential oils at concentrations far less than the 2% maximum by weight obtained by steam distillation extraction. These concentrations did not elicit a hypersensitivity response.

Degradation of TNT by bovine rumen fluid, a novel source of anaerobic microbes, was investigated. Whole rumen fluid contents were spiked with TNT and incubated for a 24 h time period. Supernatant samples taken at 0, 1, 2, 4, and 24 h were analyzed by reverse-phase HPLC with diode array detection. Within 1 h, TNT was not detectable and reduction products of TNT including 2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were present with smaller amounts of diamino-nitrotoluenes. Within 2 h, only the diamino and dihydroxyamino-nitrotoluene products remained. After 4 h, 2,4-diamino-6-nitrotoluene and 2,4-dihydroxyamino-6-nitrotoluene were the only known molecular species left. At 24 h known UV absorbing metabolites were no longer detected, suggesting further transformation such as complete reduction to triaminotoluene or destruction of the aromatic ring of TNT may have occurred. TNT was not transformed at 24 h in autoclaved and buffered controls. This study presents the first direct evidence of biodegradation of TNT by ruminal microbes.

A mixed culture of ovine ruminal microbes metabolizes the macrocyclic pyrrolizidine alkaloids present in the plant Senecio jacobaea , including jacobine and seneciphylline. Previous attempts to identify metabolites of these alkaloids have not been successful. The objective of this study was to compare the metabolism of pyrrolizidine alkaloids by a mixed culture of ovine ruminal microbes to the metabolism of pyrrolizidine alkaloids by the known organism Peptostreptococcus heliotrinreducens . P. heliotrinreducens metabolizes the pyrrolizidine alkaloids heliotrine and lasiocarpine to 7α-hydroxy-1-methylene-8α-pyrrolizidine and 7α-angelyl-1-methylene-8α-pyrrolizidine, respectively. This organism does not metabolize the pyrrolizidine alkaloids jacobine or seneciphylline. A mixed culture of ovine ruminal microbes also metabolized heliotrine and lasiocarpine to identical methylene compounds. This mixed culture also metabolized jacobine and seneciphylline, with the production of very low levels of the corresponding 1-methylene compounds. Samples were analyzed by TLC and GC/MS.

Ergot alkaloids present in endophyte-infected (E+) tall fescue cause fescue toxicosis and other toxic effects in livestock that consume infected plant tissue, leading to significant financial losses in livestock production each year. The predominant method currently in use for quantifying ergot alkaloid content in plant tissue is through high-performance liquid chromatography (HPLC), which quantifies the amount of ergovaline, one of many ergot alkaloids in E+ plant tissue. The enzyme-linked immunosorbent assay (ELISA) method used in this study detects quantities of nonspecific ergot alkaloids and therefore accounts for greater amounts of the total ergot alkaloid content in E+ tissue than does HPLC. The ELISA can also be used to more expediently analyze a larger number of forage samples without sophisticated and costly analytical equipment and therefore could be more desirable in a diagnostic setting. The purpose of this study was to evaluate the between-day and within-run variability of the ELISA and to determine the binding efficiency of 6 ergot alkaloids to the 15F3. E5 antibody used in the competitive ELISA to ascertain its feasibility as a quick analysis tool for ergot alkaloids. Straw samples had an average coefficient of variation (CV) for concentration of 10.2% within runs and 18.4% between runs, and the seed samples had an average CV for concentration of 13.3% within runs and 24.5% between runs. The grass tissue-based lysergic acid standard curve calculated from the ELISA had an average r2 of 0.99, with a CV of 2.1%. Ergocryptine, ergocristine, ergocornine, and ergotamine tartrate did not bind strongly to the 15F3.E5 antibody because of the presence of large side groups on these molecules, which block their binding to the antibody, whereas ergonovine and ergonovine maleate were bound much more efficiently because of their structural similarity to lysergic acid. Clarified rumen fluid was tested as an additional matrix for use in the ergot alkaloid competitive ELISA to determine whether future livestock metabolism experiments on the postingestion fate of ergot alkaloids in ruminants could utilize this assay as a quick screening tool for the presence of nonspecific ergot alkaloids in rumen fluid. HPLC and ELISA procedures were compared for their ability in determining ergot alkaloid toxicity based on the repeatability of the procedures and on the specific compounds they measure. The ratio of ELISA concentration to HPLC concentration (ergovaline) varied from 2.00 to 2.81 in seed samples and from 0.62 to 8.66 in straw samples, showing no consistent pattern between the 2 methods. Based on the lack of data at present for the identity of the toxin causing endophyte toxicosis and the lack of agreement between the ergovaline HPLC and ELISA analyses for ergot alkaloids, each method is equally valid as an indicator of toxicity and is the best means for determining the quantity of the specific toxin(s) they measure.

PIERCY, R. J., K. W. HINCHCLIFF, P. S. MORLEY, R. A. DISILVESTRO, G. A. REINHART, S. L. NELSON, JR., K. E. SCHMIDT, and A. M. CRAIG. Association between vitamin E and enhanced athletic performance in sled dogs. Med. Sci. Sports Exerc., Vol. 33, No. 5, 2001, pp. 826-833. Purpose: To determine the association between prerace plasma vitamin E concentration and performance in sled dogs competing in the 1998 Iditarod Race. Methods: Prerace blood samples were collected from 670 dogs. Samples were analyzed for plasma vitamin E concentration while controlling for selected hematological and biochemical variables and signalment. Starting in teams of 16, exercise consisted of running up to 1159 miles pulling a laden sled and musher via checkpoints. The records of dogs that were withdrawn from the race for health reasons, fatigue, or strategic or technical reasons, and those of dogs that finished the race were analyzed. Multiple logistic regression and Cox proportional hazards analysis were used to determine factors associated with endurance. Multiple linear regression analysis was used to determine factors associated with team speed. Results: A total of 323 dogs (48%) were withdrawn from racing at various distances from the start. Median time to finish for 39 teams was 11.5 d d and the winning time was 9.2 d. Dogs with prerace plasma vitamin E concentrations > 40.7 μg.mL -1 were 1.9 times more likely to finish (P = 0.0006) and had 1.8 times less of a risk of being withdrawn for every mile ran (P = 0.03) than were dogs with plasma vitamin E concentrations between 16.3 and 40.7 μg.mL -1 . Neither a team's mean prerace vitamin E concentration, nor the proportion of dogs within a team with high (>40.7 μg.mL -1 ) vitamin E concentration was associated with team speed. Conclusions: Dogs with higher plasma vitamin E concentrations have enhanced endurance compared with dogs with lower plasma vitamin E concentrations, but the plasma vitamin E status of a team is not associated with team speed.

Exertional rhabdomyolysis (ER) is common in sled dogs, animals with high energy expenditures that consume high fat (60% of ingested calories) diets. Associations between pre-race plasma [vitamin E] and total antioxidant status (TAS) and risk of developing ER were examined in dogs competing in the 1998 Iditarod race. Pre-race blood samples were collected from 750 dogs and a second sample was collected from 158 dogs withdrawn from the race at various times. Plasma creatine kinase activity was used to identify withdrawn dogs with ER. There was no association between pre-race plasma [vitamin E] and risk of development of ER. Dogs that developed ER started the race with higher TAS, but when withdrawn, had lower TAS than unaffected dogs and had similar pre-race [vitamin E] but higher [vitamin E] at time of withdrawal. Hence, the risk of ER in sled dogs is not affected by plasma [vitamin E] before the race.

Sodium cyanate, a neurotoxic chemical in rodents, primates and humans, is implicated in neurodegenerative disorders in protein-deficient populations subsisting in parts of Africa on the cyanogenic plant cassava. The molecular and cellular mechanisms of cyanate neurotoxicity are not understood. This study investigates the effect of sodium cyanate on glutathione (GSH) homeostasis in rodent brain and liver in vitro and in vivo. GSH levels in mouse brain were rapidly, time- and dose-dependently decreased following intraperitoneal administration of 100, 200 or 300 mg/kg sodium cyanate. By contrast, GSH disulfide (GSSG) levels were increased and GSH/GSSG ratios were decreased in a dose-dependent manner in rat brain. Sodium cyanate depleted GSH levels in all regions of mouse brain. Brain glutathione reductase activity was dose-dependently inhibited, while glutathione peroxidase activity was not affected by sodium cyanate. The disruption of GSH homeotasis, as evidenced by reduced tissue GSH/GSSG ratios, likely results from cyanate-induced inhibition of glutathione reductase activity. The results of this study suggest that cyanate neurotoxicity, and perhaps cassava-associated neurodegenerative diseases, are mediated in part by disruption of glutathione homeostasis in neural tissue.

Ergovaline, the main ergopeptine alkaloid produced in tall fescue infected with Acremonium coenophialum, is known to cause tall fescue toxicosis. Current methods in use for quantifying ergovaline in plant material have several disadvantages, including large solvent volumes and long analysis time. We report here improvements in extraction and cleanup and the high-pressure liquid chromatographic methods. Our improvements include a 24-hour extraction time, the use of smaller solvent volumes during sample preparation, and fast analysis on the polymeric reverse-phase column. In addition to allowing the analysis of large batches to assist practitioners in the accurate diagnosis of fescue toxicosis, our method is also easily modified for other matrices, such as rumen fluid.

Selected serum constituents were analyzed from 50 adult mallards (Anas platyrhynchos) of both sexes during several stages of reproduction: pre-egg laying, egg laying, incubating, molting, and postreproductive. Similar assays were conducted on sera from ducklings aged 5 to 58 days. Values for total protein (TPR), albumin (ALB), glucose (GLU), gamma-glutamyl transferase (GGT), calcium (CA), phosphorus (PHOS) and magnesium (MG) differed by sex. When all data were combined and analyzed for sex-related differences within each reproductive condition separately, all assays except lactate dehydrogenase (LD-L), cholinesterase (CHE), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRN) and direct bilirubin (BIDI) differed between sexes during one or more reproductive periods. Each assay showed differences among the various reproductive conditions regardless of gender. The pattern of change differed between sexes. All assays except ALB, GLU, CA and MG showed age-related changes. Lipemia in the sample interfered with all chemistries except TPR, LD-L and CA. Results indicate that when using clinical chemistry as a diagnostic tool in the mallard, age and reproductive condition should be determined in order to compare the data to appropriate control values.

Summary Relationships among hemoglobin (Hb) types and production traits were examined in 294 crossbred ewes from North Country Cheviot, Dorset, Finnsheep and Romney rams, and Suffolk and Columbia-type ewes. Hb BB ewe lambs were youngest at first estrus, while Hb AA ewe lambs were oldest. Ewe lamb fertility was similar for Hb AB and Hb BB ewes, and both were superior to Hb AA ewes. When Finnsheep crossbreds were excluded, frown which Hb BB was absent, Hb BB ewes had higher ewe lamb fertility than did Hb AB ewes. There was a minor advantage of the A over the B allele in ewe lamb prolificacy. Hb BB ewes were highest, Hb AB ewes were intermediate and Hb AA ewes were lowest for average fertility, prolificacy per ewe lambing, lambs born per ewe exposed to mating, total number of lambs weaned and total kilograms of lamb weaned. Grease wool production, staple length, fiber diameter grade, incidence of medullated fibers and incidence of cotted fleeces all were similar across Hb types. Likewise, Hb type did not affect milk production or composition. Ewes with Hb AB had the lowest incidence of footrot; Hb AA and Hb BB ewes were similar. Ewes with Hb AA had the lowest fecal parasite egg counts, while ewes with Hb AB or Hb BB were similar. Ewes with Hb AA also had the lowest incidence of mastitis, Hb BB ewes were intermediate and Hb AB ewes were highest. The combination of overdominance (as for footrot resistance), beneficial effects of the A allele on health-related traits (as for mastitis and parasite resistance) and the beneficial effect of the B allele on reproductive traits, if real, could partly explain the genetic polymorphism commonly reported at the Hb locus in sheep.

The intercalation model of DNA–hydrocarbon binding appears reasonable, but rests on indirect evidence only. To test the model, a size criterion for binding has been proposed. The size criterion is based on the assumption that hydrophobic forces play a major role in the binding of hydrocarbons to DNA. It states that hydrocarbons which are small enough to intercalate into DNA and be well protected from contact with the medium, will be found to bind to DNA; those that are too large will not. We report results on the binding of fourteen polycyclic aromatic hydrocarbons to DNA. Predictions based on the size criterion were found to be valid in all cases tested.

A number of polycyclic aromatic hydrocarbons bind to the double-stranded, acid form of polyadenylic acid (poly A). Model building shows that these hydrocarbons may intercalate in the helix, and be well protected from contact with the aqueous medium. Hydrocarbons that are too large to be so protected are found not to bind. A size criterion for the binding of hydrocarbons to poly A therefore exists. This criterion differs from one that was previously found for DNA. The size criteria for DNA and poly A, together, serve as strong evidence for the intercalation model for hydrocarbon complexes. Model-building experiments show that only a small portion of the hydrocarbon need extend into the medium to prevent binding. This finding implies that in two cases (1,2,5,6-dibenzanthracene·poly A and 3,4-benzpyrene·DNA) the structure of the complex is almost completely determined by the size criterion alone.

Ultraviolet absorption, fluorescence excitation and emission, and fluorescence anisotropy demonstrate that the polycyclic aromatic hydrocarbone 3,4-benzpyrene, Pyrene, anthracene, and 9-methylanthracene bind to histone IV after the sow conformational change in the presence of divalent phosphate anion. These hydrocarbons do not bind, within our limits of detection, to histone IV in the diosorderde form at low pH or to the form produced by the fast conformational change when the slow change is prevented. High anisotropy values for the bound hydrocarbon indicate a high degree of aggregation for the histone IV.

Synthetic vitamin E, all-rac-alpha-tocopherol, contains eight different stereoisomers, only one of which, RRR-alpha-tocopherol, occurs naturally. The objective of this investigation was to evaluate the relative abilities of these two vitamin E forms to enrich piglet tissues when fed as alpha-tocopheryl acetates to sows during pregnancy and lactation. alpha-Tocopherol delivery to fetuses and to suckling piglets was monitored by feeding 150 mg each of d(3)-RRR-alpha- and d(6)-all-rac-alpha-tocopheryl acetate to three pregnant sows daily from 7 d before to 7 d after giving birth. Labeled and unlabeled vitamin E concentration ratios were measured in sow plasma and milk, and in piglet (n = 9) plasma and tissues at birth, 7 and 21 d. At birth, despite elevated sow plasma deuterated alpha-tocopherol concentrations, no labeled alpha-tocopherol was detected in piglet plasma or tissues. Sow plasma and milk d(3)-alpha- to d(6)-alpha-tocopherol concentrations were 2:1, leading to a 2:1 ratio in suckling piglet plasma and tissues. In piglets at d 7 compared with birth, most tissues contained a 10-fold increase in total alpha-tocopherol; the highest deuterated vitamin E concentrations were in the liver, followed by the lung, heart, kidney, muscle, intestine and brain. In conclusion, pigs discriminate between RRR- and all-rac-alpha-tocopherol with a preference for RRR-alpha-tocopherol; thus, the official bioequivalence ratio of 1.36:1 RRR- to all-rac-alpha-tocopherol is underestimated. After initiation of suckling, piglet plasma and tissues demonstrated a dramatic increase in vitamin E concentrations, emphasizing the limited placental vitamin E transfer and the importance of milk for enhancing the vitamin E status of the newborn.